The present invention relates to a method for preparing a template DNA from a processed food using seeds of a plant such as soybean as a material, the template DNA being usable for detecting the genes of the plant and recombinant genes thereof by a PCR method and to a method for detecting the genes of a plant using such a template DNA in detecting the genes of the plant in the processed vegetable food derived from the plant by a PCR method.
Processed food using seeds of a plant as a material are subject to high temperature treatment, high temperature grinding treatment, high pressure treatment, or mechanical grinding treatment during the process of its production and as a result fragmentation of DNA molecule occurs. In the case of fermented foods such as bean paste and fermented soybeans, further fragmentation of DNA occurs due to nuclease originated from microorganisms.
The present invention is to provide a method for preparing a DNA fragment having a purity capable of using as a template for PCR amplification from such a processed vegetable food. Further, the present invention is to provide a method for using said template DNA in detecting by a PCR method the genes of a plant from which processed vegetable food is produced.
For the extraction of plant DNAs, i.e., DNAs of soybean and other plants, there have already been developed fundamental techniques, which can also be used for the extraction of DNA from foods, such as the CTAB method (Marray, M. G. and Thompson, W. F. (1980); Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res., 8, 4321-4325), the SDS-phenol method (David, R. W., Thomas, M., Cameron, J., St. John, T. P., Scherer, S. and Padgett, R. A.; (1980) xe2x80x9cMethods in Enzymologyxe2x80x9d, ed. by Grossman, L. and Moldav, K., Vol. 65, p404, Academic Press, New York), and the protease K method (Jofuku, K. D., Goldberg, R. B.; (1988) Plant molecular biologyxe2x80x94a practical approach, ed. by Show, C. H., p37, IRL Press, Oxford-Washington D.C.).
However, when processed foods using plant seeds as a material are produced, generally the material is often subjected to various processing steps such as high temperature, high pressure, and mechanical grinding treatments, or fermentation operation with microorganisms. For this reason, if the above fundamental techniques are applied directly, it has been in many cases difficult to prepare a DNA having a sufficient purity to be used as a template DNA in PCR amplification.
For example, in the case of soybean, which is one of materials for Japanese traditional plant-derived foods, seeds and bean sprouts allow extraction of high purity DNA which is in a native state only by use of the above fundamental methods. Tofu (bean curd) inclusive of kori-dofu (freeze-dried tofu) and aburaage (fried tofu slice) inclusive of ganmodoki (fried stuffed tofu), etc. can be used for preparing a template DNA for PCR by use of the above fundamental methods without pretreatment or DNA purification operation since they have undergone DNA fragmentation during their production, although yield of the template DNA is low.
In the case of kinako (parched bean flour) (high temperature grinding) and steamed/boiled soybean (cooked bean, autoclaving), or their fermented foods (natto (fermented soybeans), miso (bean paste), etc.) and the like, the DNA is cut into fragments by the respective treatment involved and hence it has been very difficult to prepare high purity DNA with which no low molecular weight DNA is mixed.
The first object of the present invention is to provide a method for preparing high purity DNA which can be used as a template for PCR from processed vegetable foods derived from soybean and other plant seeds as a material, the processed foods having been subjected to denaturation treatment for protein.
Another object of the present invention is to provide a method for detecting a gene of a plant by a PCR method from a processed vegetable food derived from the plant, using said template DNA.
To know exactly what the materials contained in a highly processed food are like is now a global trend in the stream of consumers"" consciousness. However, many of the operations during the processing procedure are often accompanied by considerable denaturation of proteins so that the identification of plant materials by the analysis of proteins according to the conventional methods were successful in only limited situations.
In order to obviate the above problems, the present inventors have made intensive research and as result they have found that use of optional pretreatment prior to fundamental methods for DNA extraction and application of specified purification and separation methods after the extraction enable preparation of high purity DNA even from processed vegetable foods which has been subjected to a protein denaturation treatment.
Thus, if DNA which is resistant to heat treatment and the like, that is, remains in the processed food in an uncleaved state after the protein denaturation treatment can be prepared in a highly pure state, then it is possible to clarify the name of the plant material with high sensitivity by detecting the gene of the plant by a PCR method using this. Finally, there is the possibility that a judgment can be realized as to whether or not the plant as a raw material of processed vegetable food is a plant that has been subjected to gene recombination treatment. The present invention has been achieved based on these findings.
Therefore, according to a first aspect of the present invention, there is provided a method for preparing a template DNA from a processed vegetable food, comprising the steps of:
extracting a crude DNA fraction from a processed vegetable food subjected to at least one of a high temperature treatment, a high temperature grinding treatment, a high pressure treatment and a fermentation treatment, and optionally subjected to defatting; and performing a DNA fractionation treatment of the crude DNA fraction by a polyethylene glycol precipitation method and/or a polyacrylamide gel electrophoresis method.
According to a second aspect of the present invention, there is provided a method for detecting a gene of a plant in a processed vegetable food derived from the plant by a PCR method, wherein the template DNA obtained by the method of the first aspect of the present invention is used.
According to a third aspect of the present invention, in the method of the second aspect of the invention, the processed vegetable food is an oil source seed.